The degradation of most cellular proteins is regulated by coordinated addition and removal of ubiquitin by families of ubiquitin E3 ligases and deubiquitylating enzymes (DUBs) respectively. DUBs proteolytically cleave ubiquitin molecules from proteins resulting in modifications of protein activity, localization and function. Several DUBs are aberrantly regulated in cancer, including the best studied, USP7, selective inhibitors of which are active in cancer models. A second DUB, USP22, is another validated anticancer target, being one of 11 genes in the death-from-cancer gene signature, a component of the human SAGA transcriptional cofactor complex regulating myc transcription, and a regulator of the expression of p21, the histone deacetylase Sirt 1, and p53 activity. USP22 is overexpressed in oral squamous cell carcinoma, breast, non-small cell lung, colorectal, and other cancers and its expression is inversely correlated with survival. Unlike most other DUBs, USP22 exhibits robust activity only as a component of a multi-subunit complex. Initial studies reported activity solely as a member of the 2MDa SAGA complex, but more recently it has been demonstrated that USP22 exhibits similar activity in a four-protein DUB module derived from the SAGA complex. Based on Progenra's success working with DUB complexes, it is proposed here to develop a HTS compliant assay to enable the identification of novel inhibitors of USP22. Following a screen of Progenra's 200K member diversity based library of small molecules, compounds of interest will be characterized further against a panel of DUBs and other proteases and cellular activity will be explored by testing the modulation of a series of well validated pharmacodynamic markers. The primary goal is to identify novel inhibitors of USP22; specific milestones include: purification of active USP22 DUB complex, configuration of an HTS compliant USP22 assay format and identification of selective USP22 inhibitors with activity in cellular models. The most interesting compounds will be advanced, in Phase II, to hit-to-lead medicinal chemistry optimization with associated DMPK and in vivo activity.